This invention relates to an electrode having a coating on an electroconductive core, the coating comprising at least two layers with at least one of the layers being a polymer network cross-linked by high energy radiation or a polymer network produced by end linking functionally terminated chains, or a polymer network produced by crosslinking chains having reactive functional groups as side chains. The electrode of the invention has particular utility as a sensor for molecular oxygen, or as an amperometric sensor having an enzyme or antibody immobilized in the cross-linked polymer layer.
The method of producing an electrode in accordance with the invention is simple and inexpensive in comparison to prior art practices. Moreover, the method is adapted to the fabrication of miniaturized electrodes, which is a feature generally recognized as being desirable but not hitherto attainable.
An oxygen sensor electrode of the invention exhibits enhanced response time as compared to presently available electrodes. An amperometric sensor electrode in accordance with the invention, when used as an electroanalytical device, exhibits enhanced response time, selectivity and sensitivity, i.e. low detection limits.
The Clark electrode, disclosed in U.S. Pat. Nos. 3,380,905; 3,539,455; and 3,912,386, is widely used for the determination of oxygen. It comprises a conducting metal such as platinum and a reference electrode mounted behind a thin membrane with a thin layer of electrolyte sandwiched between the membrane and the electrodes. Oxygen in a sample to be analyzed diffuses through the membrane and electrolyte layer to the metal electrode where it is detected by reduction. The membrane is critical in providing selectivity for oxygen by allowing only gases to pass therethrough, and it protects the electrode surface against fouling by preventing surfactants and other adsorbing species from reaching the electrode surface. The membrane may be polytetrafluoroethylene, silicone, methyl methacrylate, or cellulose acetate. Although highly successful, the Clark electrode has the disadvantages of relatively slow response time (due to the distance through the membrane and quiescent electrolyte layer which the oxygen must traverse to reach the electrode surface), and difficulty in miniaturization using conventional membrane-spacer construction.
L. C. Clark, Jr. et al, in "Rapid Micromeasurement of Lactate in Whole Blood", Critical Care Medicine, 12(5), 461-464 (1984) describe a lactate sensor comprising a very thin layer of lactate oxidase (immobilized by glutaraldehyde) held between a cellulose ester membrane and a polycarbonate membrane, cemented in an O-ring and held in place against an electrode tip.
Cross-linking of a polymer by radiation, such as ultra-violet radiation, gamma ray irradiation and the like, is well known and is disclosed, e.g., by E. S. Decastro et al in "Electrodes Coated With Polymer Networks Cross-Linked By .gamma.-Irradiation" J. Electroanal. Chem. 138, 197-200 (1982).
In the Decastro et al article gamma irradiation of platinum electrodes coated with diallyl dimethyl ammonium chloride (DDAC) and 2,6-dichlorophenolindophenol (DCIP) is disclosed. Cross-linking of electroactive DCIP into an inert DDAC network was shown by cyclic voltammagrams.
Additionally, five articles disclosing entrapment of an enzyme by irradiation have been authored by H. Maeda et al, four being published in Biotechnology And Bioengineering, John Wiley and Sons, Inc., publisher:
Maeda I "Preparation Of Immobilized Enzymes Using Poly (Vinyl Alcohol)" Vol., XV 607-609 (1973); Maeda II "Preparation Of Immobilized Enzymes By Electron-Beam Irradiation", Vol. XV 827-829 (1973); Maeda III "Preparation Of Immobilized Enzymes By N-Vinylpyrrolidone And The General Properties Of The Glucoamylase Gel", Vol. XVI 1517-1528 (1974); Maeda IV "Preparation Of Immobilized .beta.-Galactosidase By Poly (vinyl Pyrrolidone) And The Continuous Hydrolysis Of Lactose In Acid Whey", Vol. XVII 1571-1589 (1975). Maeda V is entitled "Preparation Of Immobilized Enyzmes By .gamma.-Ray Irradiation", and published in Biochimica et Biophysica Acta, 315, 18-21 (1973).
In Maeda I immobilization of glucoamylase and invertase in polyvinyl alcohol is disclosed, using gamma ray irradiation of than 4M rad for cross-linking. However, 75-80% of the enzyme activity of glucoamylase and 80-90% of the enzyme activity of invertase were lost. There is no suggestion of utilizing this procedure for an electrode.
In Maeda II immobilization of glucoamylase and invertase in polyvinyl alcohol is disclosed, using electron beam irradiation in a nitrogen atmosphere. Over 50% of the enzyme activity was lost on entrapment by electron beam irradiation with 6 Mrad. As the radiation level was increased, enzyme activity decreased substantially. At 36 Mrad there was almost no enzyme activity.
In Maeda III immobilization of glucoamylase, invertase and .beta.-galactosidase in vinyl pyrrolidone is disclosed, using gamma ray irradiation. Over 90% of enzyme activity was lost with invertase and .beta.-galactosidase, while 55% activity was lost with glucoamylase.
In Maeda IV immobilization of .beta.-galactosidase in polyvinyl pyrrolidone is disclosed using gamma ray irradiation. At radiation of 3.0 Mrad the activity of the enzyme was about 30%. Leakage of enzyme from the gel was detected where the PVP-enzyme solution contained more than 1% of enzyme protein.
In Maeda V the immobilization of enzymes by gamma ray irradiation is disclosed. Rigid gels were prepared from acrylamide monomer, eliminating the need for a cross-linking reagent. Irradiation of invertase and .beta.-galactosidase with 1 Mrad of gamma ray irradiation resulted in loss of 65-75% of enzyme activity. Irradiation of glucoamylase with 2M rad of gamma ray irradiation resulted in a 60% loss of enzyme activity.
U.S. Pat. No. 3,940,667 issued Feb. 24, 1976 to G. R. Pearce, discloses an electrode with a polymer containing coating made by applying radiation curable polymer containing material to a metal containing electrode, irradiating the applied polymer containing material by ultraviolet or electron beam radiation, and introducing an electrolyte into the cured polymer.
U.S. Pat. No. 4,579,642, issued Apr. 1, 1986 to Y. Niiyama et al, discloses an electrochemical sensor comprising a vessel with a liquid-junction at an end face, a resilient ion selective membrane at the liquid-junction, and an immobilized enzyme membrane covering the ion selective membrane.
U.S. Pat. Nos. 4,404,066 issued Sept. 13, 1983 and 4,356,074 issued Oct. 26, 1982 to J. M. Johnson and European patent application No. 104,935 published Apr. 4, 1984 disclose an electrochemical cell laminate having exterior membrane layers and an interior enzyme layer. An electrode located within the enzyme layer applies an electrical potential to the enzyme.
U.S. Pat. No. 4,444,878 issued Apr. 24, 1984 to H. P. Paulus discloses an electrode, and a membrane thereon having associated therewith a plurality of molecules of each enzyme which acts on the substance to be measured and a plurality of bispecific antibody determinants bonded to the molecules of each enzyme.
Despite the considerable amount of work devoted to development of electroanalytical devices which include electrodes having an electrolyte or enzyme layer, there still exists a genuine need for an electrode, and a simple and inexpensive method for making it, which exhibits improved response time and selectivity, and low detection limits, and which can be readily miniaturized.